The filoviruses (Ebola and Marburg) cause severe hemorrhagic fever syndromes with high mortality rates. As these RNA viruses are classified as NIAID Category A pathogens, the current lack of effective vaccines and antiviral therapeutics for these deadly pathogens is particularly concerning. We and others have established that efficient filovirus budding is critically dependent on the subversion of host proteins Tsg101 and Nedd4 and that viral PTAP and PPxY late (L) budding domains are critical for these interactions, respectively. As disruption of virus budding would prevent virus dissemination, we propose to directly evaluate the ability of small molecule inhibitors to disrupt Tsg101-PTAP and Nedd4-PPxY interactions, thereby preventing virus budding. Our collaborators, Drs. Michael Lee and Mark Olson (USAMRIID, Ft. Detrick, MD), have used the known structures of Tsg101-PTAP and Nedd4-PPxY interactions to guide the in silico selection/design of competitive interaction blockers. We are currently evaluating the ability of top candidate inhibitors to disrupt the host-dependent egress of virus particles in virus-like particle (VLP) budding assays. As Tsg101-PTAP and Nedd4-PPxY interactions are too weak and/or transient to be detected by standard biochemical approaches, our laboratory has successfully developed a powerful bimolecular complementation (BiMC) approach to detect these viral-host interactions in live cells. We will use this innovative technique to determine whether candidate inhibitors disrupt these virus-host interactions. Promising preliminary results with one lead compound (5539- 0062) support the feasibility of this proposal. As L-domain containing matrix proteins are required for efficient virus-cell separation of many RNA viruses, including retroviruses, arenaviruses, rhabdoviruses, paramyxoviruses, henipaviruses, and filoviruses, we predict that targeting the interaction domain between filovirus VP40 and host Tsg101 and Nedd4 will serve as basis for the development of new and powerful broad- spectrum antiviral drugs. Our group of highly interactive and expert collaborators will build upon our promising preliminary results obtained following an in silico screen of 4.8 million compounds to functionally validate candidate small molecules that inhibit filovirus-Tsg101 (Aim 1) and filovirus-Nedd4 (Aim 2) interactions during the R21 phase of this proposal. In the R33 phase of this proposal, we will develop and utilize Fluorescence Lifetime Imaging Microscopy (FLIM) to functionally/mechanistically categorize inhibitors as they disrupt virus- host protein interactions in real time and assess their mode of action, thereby laying the foundation for development and testing of multi-drug therapies (Aim 3). Lastly, we will determine the efficacy of inhibitors in preclinical live virus studies using WT and mutant VP40 L-domain VSV recombinants already generated (BSL- 2 setting). Moreover, in collaboration with Dr. G. Olinger (USAMRIID), we will test mature lead candidate inhibitors for their ability to block egress of live, EBOV and MARV in a BSL-4 setting (Aim 4).